Kinetic characterization and phosphoregulation of the Francisella tularensis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase)

Abstract

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K(M)(DXP) = 104 microM, K(M)(NADPH) = 13 microM, k(cat)(DXP) = 2 s(-1), k(cat)(NADPH) = 1.3 s(-1)), an IC(50) for fosmidomycin of 247 nM, and a K(i) for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg(+2) as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.

Figure 1. The two isoprene biosynthetic pathways.

A) The MVA Pathway. The first two enzymes of the MVA pathway condense 3 molecules of acetyl-CoA (1) to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) (3), which is subsequently reduced to MVA (4) by HMG-CoA reductase , . MVA is phosphorylated twice then decarboxylated to yield IPP (7) –, which is converted to DMAPP (8) by an isomerase . B) The MEP Pathway. Condensation of pyruvate (9) with glyceraldehyde 3-phosphate (10) yields 1-deoxy-D-xylulose 5-phosphate (DXP; (11)) , an intermediate with a role in E. coli vitamin B1 and B6 biosynthesis – and isoprene biosynthesis. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (also called MEP synthase or IspC) catalyzes the reduction and rearrangement of 11 to yield MEP (12) , the first committed step in the E. coli MEP pathway. The next enzyme, CDP-ME synthase, converts MEP into 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME; (13)). CDP-ME kinase then phosphorylates CDP-ME, which is subsequently cyclized (coupled with the loss of CMP) by cMEPP synthase to yield 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (15) –. A reductive ring opening of 15 produces 1-hydroxy-2-methyl-2-butenyl diphosphate (HMBPP; (16)) , –, which is subsequently reduced to both IPP and DMAP in a ∼5:1 ratio , –. C) The reaction catalyzed by MEP synthase. Isomerization via cleavage of the bond between C3 and C4 and formation of a new bond between C2 and C4 produces the intermediate 2-C-methyl-D-erythrose 4-phosphate (18) , , which is subsequently reduced to yield MEP (12).

Figure 2. Dose-response plot of F. tularensis subsp. novicida growth as a function of fosmidomycin concentration.

Fractional growth was calculated as the ratio of cell density (OD₅₉₅) in the presence of inhibitor to cell density in the absence of inhibitor. Nonlinear regression fitting indicates half maximal activity at 12 µM. The goodness-of-fit (R²) value is indicated. Growth curves are presented in supportive Fig. S1.

Figure 3. Purification of recombinant F. tularensis MEP synthase.

A) Coomassie stained SDS-PAGE showing a molecular weight marker (MW) and purified His-tagged MEP synthase. His-tagged MEP synthase has a predicted molecular weight of 46.4 kDa. B) Western blot hybridization analysis of purified MEP synthase using an anti-His antibody results in an intense band of the expected size. The appearance of a weak, smaller molecular weight band suggests that some degradation may have occurred.

Figure 4. The substrate dependent activity of F. tularensis MEP synthase.

Michaelis-Menten plots of reaction velocity as a function of A) DXP concentration and B) NADPH concentration were used to derive the kinetic parameters listed in Table 1. The solid line represents the nonlinear least-squares best fit of the data to the Michaelis-Menten equation. The R² value for each plot is indicated.

Figure 5. The divalent cation specificity of F. tularensis MEP synthase.

Enzyme assays were performed with several different divalent cations at a fixed DXP (150 µM) and NADPH (100 µM) concentration. Relative enzyme activity reveals the preference of the enzyme for Mg⁺².

Figure 6. Graphical determination of the inhibition constant.

Because fosmidomycin is a slow, tight binding inhibitor , the enzyme was preincubated with fosmidomycin for 10 minutes prior to addition of substrate. The absolute value of the x intercept of the line produced from linear regression fitting the plot of K _M ^app,DXP as a function of fosmidomycin concentration defined the K_i as 98.9 +/− 4.5 nM. The R² value is indicated.

Figure 7. Predicted tertiary structure of F. tularensis MEP synthase, homology-modeled using SWISS-MODEL.

A crystal structure of F. tularensis MEP synthase has not been reported. To permit the visualization of phosphoserine177 within the context of the tertiary structure, the F. tularensis MEP synthase was modeled based upon the resolved structure of the E. coli homolog (48% identity, 66% homology). A cartoon representation of the model is shown, with alpha helices colored red, beta sheets colored yellow, and coiled regions colored gray. The beta sheets comprise the dimer interface in the E. coli structure. The quality of the model was evaluated with ProQRes (Fig. S3) which provides scores ranging from 0 (unreliable) to 1 (reliable). Regions of the model scoring <0.5 are colored light blue. Primary sequence alignment and the structure of E. coli MEP synthase were used to identify residues comprising the substrate binding site (colored dark blue with backbone and sidechain residues shown). Serine177 (colored green with backbone and sidechain residues shown) is equivalent to E. coli Ser186, which contributes to the substrate binding site and has been shown to participate in conformational changes that occur upon substrate binding . Two tryptophan residues present in the F. tularensis MEP synthase model are colored pink.

Figure 8. Regulation of F. tularensis MEP synthase.

A) Intrinsic fluorescence spectra of MEP synthase and its mutants. Wildtype and mutant (S177D and S177E) proteins were adjusted to 5 µM in 0.1 M Tris pH 7.5, 1 mM NaCl, 5 mM DTT and analyzed using an excitation wavelength of 290 nm. The emission spectra was measured from 310 to 400 nm. The Em λ_max of wildtype MEP synthase was detected at 335 nm, of S177E was detected at 337 nm, and of S177D was detected at 326 nm. The blue shift observed with S177D is indicative of a conformational change sequestering tryptophan residues into a hydrophobic environment. The slight red shift observed with S177E is indicative of a conformational change exposing tryptophan residues to a hydrophilic environment. The increased quantum yield observed with both S177D and S177E is also indicative of a structural change in MEP synthase. B) The relative catalytic activity of wildtype MEP synthase and the S177D and S177E mutants. Assays were performed with 300 µM DXP and 150 µM NADPH.